Project Summary. Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents off-pathway interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of virulence-associated surface structures by a broad range of pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. The goals of this proposal are to probe the structure and function of the usher to gain an understanding of the molecular mechanisms governing pilus biogenesis across the OM and to use the chaperone/usher pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the following hypotheses: The usher forms a twin-pore complex in the OM that functions as a pilus assembly and secretion site. Distinct domains of the usher function to control, coordinate, and catalyze the exchange of chaperone-subunit interactions for subunit-subunit interactions at the periplasmic face of the usher to drive the ordered assembly and secretion of the pilus fiber through the usher to the cell surface. The first specific aim is to probe the molecular mechanisms of pilus biogenesis at the usher. The second specific aim is to analyze usher-chaperone-subunit interactions both in vivo and in vitro. The third specific aim is to determine the structure of the usher using X-ray and electron crystallography, and image pilus assembly intermediates using cryo-electron microscopy. Relevance. The work described in this proposal will elucidate mechanisms of organelle biogenesis and virulence factor secretion by pathogenic bacteria. Knowledge gained from this proposal will create opportunities for the development of novel antimicrobial agents, urgently needed during this time of increasing antibiotic resistance.